Toxoplasma gondii (Tg) is an obligate intracellular category B priority pathogen that serves as a model system for other priority B protozoa including Cryptosporidium parvum and Cyclospora cayetanensis. Tg is the causative agent of toxoplasmosis, a known health hazard in immunocompromised individuals including transplant recipients. Our overall goal is to determine mechanisms used by Tg to protect itself against cell autonomous immunity. Mechanisms Tg has evolved to protect itself within its host cell likely underlie its ability to use phagocytes to disseminate to the brain and CNS to survive to establish chronic infection and to persist within cysts long term to enable reactivation of disease. In previous studies we used an unbiased approach to let Tg identify for us the defenses it has evolved to protect itself from cell autonomous immunity. Unexpectedly, we isolated a panel of independently- derived Tg mutants that all share a pronounced inability to withstand nitrosative stress (RNS) characterized by fragmentation of the parasites' sole mitochondrion suggestive of RNS-induced parasite autophagy resulting in parasite stasis or degradation. The mutants are also impaired for their ability to establish/maintain chronic infection in vivo. The goal of this R3 proposal is to use the mutants we have isolated to identify Tg genes important for conferring resistance to RNS as well as for in vivo infection. We have identified the genomic site of plasmid insertion for four of the mutants. These four genes that appear to be unique to Tg will be deleted in wild type (WT) parasites to validate their role in parasite resistance to RNS. Whole genome mutational profiling has only recently become a productive method to identify single nucleotide polymorphisms (SNPs) and insertions/deletions in chemical and insertional Tg mutant. We will use this new innovation to identify the plasmid insertion site in seven other mutants where the primary gene disruption remains unknown. The proposal fulfills the goal of the R03 grant mechanism as the identification of the parasite genes important for resistance to RNS is a secondary analysis of the mutants identified in our previous forward genetic screen and as such is a defined extension of the original study.